The liver is an important organ in an animal which takes part in the metabolism, and the liver function is originated from hepatocytes that occupies about 70% of the liver. Such a function is effected not simply by the hepatocytes but by their interactions with non-parenchymal cells and extracellular matrix, and by the construction of tissue based on these interactions. In other words, biological activities of the liver in the living body are effected by the formation of spheroids in which hepatocytes are adhered to one another.
Previously, the inventors of the present invention have conducted studies on the method of culturing hepatocytes for formation of spheroids with retaining their functions, and discovered a substance related to the reconstitution of tissue morphology of hepatocytes capable of maintaining the liver function at a high level. Based on this finding, the inventors have also succeeded in forming spheroids of hepatocytes which can express and maintain their functions at high levels for a prolonged period of time, by culturing hepatocytes in the presence of the above-described substance, and in reproducing the tissue construction to a certain degree.
That is, as shown in FIG. 1, when hepatocytes were isolated from adult rat livers by a collagenase-liver-perfusion method, inoculated in a culture dish which has been coated with proteoglycan(s) of liver reticulin fibers and then cultured statically in serum-free hormone-defined medium (HDM) supplemented with the necessary hormones such as EGF (epidermal growth factor), insulin and the like, the inoculated hepatocytes attached to the coated substrate to form monolayers during the initial stage of the cultivation, and, as the cultivation progressed, the monolayers assembled to form multilayer islands and the multilayer islands shrunk to form spherical cell clusters which subsequently separated from the surface of the dish to form floating spheroids in the liquid medium (Cell Struct. Funct., 13, 179 (1988), Biochem. Biophys. Res. Commun., 161, 385 (1989)).
It has been revealed that the glycan moiety of the above-described reticulin-originated proteoglycan(s) consisted of dermatan sulfate, heparan sulfate and other unknown sugars. However, when culture dishes were coated with chondroitin sulfate, dermatan sulfate, heparan sulfate or an adhesive substrate such as collagen extracted from rat livers or fibronectin fractions or a glycoprotein, hepatocytes spread in the dishes but remained in the state of monolayers and did not form spheroids.
When hepatocytes are cultured in a positively charged polystyrene plastic dish, they form floating spheroids similar to the case of their cultivation in a proteoglycan-coated culture dish. It is considered that such a phenomenon occurs because hepatocytes secrete proteoglycan(s) when inoculated in this type of plastic dish and the secreted proteoglycan(s) adhere to the surface of the dish (Exp. Cell Res., 186, 227 (1990), JP-A-1-277486 (the term "JP-A" used herein means an "unexamined published Japanese patent application")).
When cultured in the presence of the proteoglycan as a culture substrate, hepatocytes form floating spheroids which are stable even after a relatively prolonged cultivation period. It has been reported that the hepatocyte spheroids could maintain liver-specific differentiation function at a high level, because they were able to secrete albumin at a high level constantly for a prolonged period of time in comparison with monolayers of hepatocytes, and that the hepatocyte spheroids possibly showed a tissue reconstruction quite close to the in vivo structure, because they hardly showed activity in cell proliferation at least when checked by .sup.3 H-thymidine incorporation and nuclear labeling index (J. Clin. Electron Microscopy, 21, 5 (1988)).
However, since the proteoglycan(s) cannot be easily prepared from reticulin fibers in high yield, great concern has been directed toward the deveolopment of a culture substrate which, as a substitute of the proteoglycan(s), can induce spheroids formation of hepatocytes efficiently and is effective for the continuative expression of hepatocyte differentiation functions. In addition, it is required to culture hepatocytes in vitro for a prolonged period of time with retaining their in vivo functions for development of a biological artificial liver function-aiding device.